In recent years, an RNA interference (RNAi) method using a 21-base-long, short double-stranded RNA (small interfering RNA: siRNA) has been attracting attention. According to this RNAi method, an approximately 100-base-pair-long double-stranded RNA is transfected into a cell so as to be digested into about 20- to about 25-base-pair-long double-stranded RNA fragments by the action of Dicer in the cytoplasm. The RNA fragments are then combined with a plurality of proteins to form an RNA/protein complex (this complex is referred to as an “RISC”: RNA-Induced Silencing Complex), which binds to a homologous region of mRNA produced from the target gene and thereby potently inhibits gene expression. The principal method used today utilizes a chemically synthesized 21-base-long double-stranded RNA having a dangling end of two bases on the 3′-end. A recent report by J. Rossi et al. revealed that a 27-base-pair-long double-stranded RNA has an RNA interference effect that is about 100 times greater than that of a 21-base-long siRNA (see Non-Patent Document 1). This great effect is considered to be achieved for the following reason: after a 27-base-pair-long RNA is cleaved with an RNase III-like enzyme, Dicer, into a 21-base-long siRNA, the siRNA is recognized as is by the protein complex RISC, allowing siRNA effects to be exhibited with high efficiency.
According to the above RNA interference method using a synthetic RNA, sample preparation is relatively easy and handling is simple. Therefore, the method has been attracting a great deal of attention in the field of biotechnology business, as well as in the life science field.
However, even this excellent RNA interference method is unsatisfactory in terms of intracellular stability, cellular uptake, intracellular localization, gene expression-inhibiting effect, target specificity, etc.; accordingly, further improvement in these aspects has been desired. Recently, various chemically modified siRNAs have been produced to provide synthetic siRNAs with enhanced nuclease resistance and highly active RNA interference effects. For example, to enhance the resistance to exonuclease digestion, siRNAs, whose end is modified with an amino group or a thiol group etc. or is modified to form an abasic site etc., have been synthesized. However, it has been reported that although terminal modification of a double-stranded RNA having an RNA interference effect may enhance nuclease resistance and increase the transfection rate, it also greatly reduces the RNA interference effect. It was thus impossible in the prior art to provide an improved double-stranded RNA having an RNA interference effect, which has an enhanced nuclease resistance and cell transfection rate as well as a further increased RNA interference effect.
β-1,3-glucan is a polysaccharide that is actually used in the form of a clinical intramuscular injectable formulation. It has long been known that natural β-1,3-glucan exists as a triple helix (see Non-Patent Document 2). In vivo safety of this polysaccharide has also been confirmed, and β-1,3-glucan has been used as an intramuscular injectable formulation for about 20 years (see Non-Patent Document 3). It has also been reported that β-1,3-glucan has drug delivery ability, and a covalent bond formed between β-1,3-glucan and the drug enables a drug to be delivered to a target site (see Patent Document 1).
Natural β-1,3-glucan is known to exist as a triple helix. Further, it has been revealed that when this polysaccharide is dissolved in a polar solvent so as to be disintegrated into independent single strands, then single-stranded nucleic acid is added, and the solvent is replaced with water (a renaturation process), a triple helical complex consisting of one strand of nucleic acid and two strands of the polysaccharide is formed (see Non-Patent Document 4). It is believed that the nucleic acid and the polysaccharide in such a triple helical complex are complexed principally by hydrogen bonds.
In recent years, the present inventors further revealed that the delivery of a gene can be accomplished by forming a complex comprising β-1,3-glucan and the gene (see Patent Document 2). Further, a method for transfecting nucleic acid using β-1,3-glucan having a cell membrane-permeable functional group and a lipid membrane disrupting functional group was reported (see Patent Document 3). However, these publications only disclose methods for transfecting a single-stranded nucleic acid into cells, and are silent as to double-stranded siRNA etc. Patent Document 4 discloses a method for transfecting a double-stranded DNA having genetic information into a target cell using β-1,3-glucan. However, putting this method to practical use appears to be difficult due to its low transfection efficiency.
Thus, when β-1,3-glucan is merely complexed with a double-stranded RNA having an RNA interference effect, a triple helical structure is not formed, or a triple helix is formed, but the double-stranded RNA may no longer be double-stranded; accordingly, the desired effect cannot be expected to be produced. Further, a double-stranded RNA having an RNA interference effect is different from a DNA having genetic information in the mechanism of action required in cells. Intracellular complexing of the double-stranded RNA with RISC, and binding to a homologous region of mRNA produced from the target gene are important to provide the RNA interference effect. Therefore, even if the method disclosed in Patent Document 4 is applied to a double-stranded RNA having an RNA interference effect, a low RNA transfection efficiency, as in the case of the DNA, or a great reduction of the RNA interference effect in cells is expected.    Patent Document 1: WO 96/014873 pamphlet    Patent Document 2: WO 01/34207 pamphlet    Patent Document 3: Japanese Unexamined Patent Publication No. 2006-69913    Patent Document 4: Japanese Unexamined Patent Publication No. 2005-204612    Non-Patent Document 1: J. Rossi et al., Nature Biotech., 23, 222-226 (2005)    Non-Patent Document 2: Theresa M. et al., J. Am. Chem. Soc., 120, 6909 (1998)    Non-Patent Document 3: Hasegawa, Oncology and Chemotherapy, 8, 225 (1992)    Non-Patent Document 4: Kazuo Sakurai, Polym. Preprints. Jpn., volume 49, page 4054, 2000